|
| Status |
Public on Jun 13, 2010 |
| Title |
E2AKO-H3K4me1-6h-bHLHER-ChIP-Seq |
| Sample type |
SRA |
| |
|
| Source name |
E2A-/- pre-pro-B cell
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: pre-pro-B cell
chip antibody: H3K4me1 (ab8895)
transgenes: bHLH-ER
genetic background: E2A-/-
|
| Treatment protocol |
The cells were spin-infected with a retrovirus expressing truncated E2A (lacking both activation domains) fused to the estrogen receptor ligand binding domain, as well as hCD25 from a second cistron. After 21 hr in culture, cells were treated with tamoxifen (1 µM final concentration) for 6 hr. Cells expressing hCD25 were then enriched magnetically (Miltenyi).
|
| Growth protocol |
E2A-/- pre-pro-B cells were cultured on S17 stromal cells in the presence of IL7, SCF, and FLT3 ligand in Iscove’s Modified Dulbecco’s medium and in the presence of 10% FCS, penicillin, streptomycin and L-glutamine.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated cells or nuclei and protein-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation, ChIP DNA was PCR-amplified with Illumina primers for 12-18 cycles and library fragments (ChIP) (insert plus adaptor and PCR primer sequences) were size selected from a 2% agarose gel or a 8% polyacrylamide gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer I or Genome Analyzer II following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Chromatin IP against H3K4me1 in E2A-/- pre-pro-B cells expressing an tamoxifen activated E2A mutatant lacking both transactivation domains (bHLH-ER) for 6hr
|
| Data processing |
Alignment: Reads were truncated to 25 bp and aligned to the mouse mm8 genome (NCBI Build 36) using eland. Only reads that mapped to a single, unique position were used for downstream analysis.
Peak Identification: Peaks were identified using custom software (HOMER, available at http://biowhat.ucsd.edu/homer/). For transcription factors, peaks were found by identifying 200 bp regions with tag density exceeding a threshold set at a 0.1% false discovery rate (determined by tag randomization). Only peaks with >4x normalized tag density relative to control sequencing and >4x normlaized tag density to the sourrounding 10kb of region were considered for analysis.
|
| |
|
| Submission date |
May 24, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Christopher Benner |
| E-mail(s) |
cbenner@ucsd.edu
|
| Organization name |
University of California, San Diego (UCSD)
|
| Department |
Medicine
|
| Street address |
9500 Gilman Dr. MC 0640
|
| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92093-0640 |
| Country |
USA |
| |
|
| Platform ID |
GPL9250 |
| Series (1) |
| GSE21978 |
A global network of transcription factors, involving E2A, EBF1 and FOXO1, that orchestrates the B cell fate |
|
| Relations |
| SRA |
SRX021886 |
| BioSample |
SAMN00014657 |
| Named Annotation |
GSM546532_Sample16.E2AKO-H3K4me1-6h-bHLHER.bed.gz |
